Pregabalin, chemically known as 3-(S)-(aminomethyl-5-methyl hexanoic acid having structure formula (1) is known to treat several central nervous system disorders that include epilepsy, neuropathic pain, anxiety and social phobia.

(S)-Pregabalin has been found to activate GAD (L-glutamic acid decarboxylase) in a dose dependent manner and promote production of GABA (gamma-aminobutyric acid), one of the major inhibitory neurotransmitters of brain. The discovery of antiseizure activity was first disclosed in U.S. Pat. No. 5,563,175.
Pregabalin has been prepared in various ways. One of the common approaches involves synthesis of racemic Pregabalin typically a 50:50 mixture of R and S isomers and subsequent resolution through diastereomeric salt formation. Such an approach could be found in Patent publications such as WO2009122215, WO2009087674, WO2009044409, WO 2008138874, WO2009125427 and WO2009001372. The major difficulties associated with this approach involve the loss of R-enantiomer along with a part of S-isomer as well and this can not be effectively recycled leading to cost pressure. Another approach has utilized resolution in the intermediate stage as a strategy. Scheme 1 outlines the approach described in WO 9638405. The synthesis involves Knovanagal condensation followed by Micheal addition and acidic hydrolysis gives diacid. The diacid was converted to mono amide which was resolved by (R)-phenylethylamine. After liberation of R-mono acid amide it was converted to (S)-Pregabalin by Hoffmann degradation. The overall yield was 12% and enantiomeric excess (ee) 99.8% over 8 steps. All commercially reagents were used and the chiral auxiliary can be recovered.

In WO2008137512, another approach described as shown in scheme 2, which involves resolution of amide intermediate followed by Hoffmann degradation.

A further modification was described in OPRD, 2009, 13, 812-13. The approach described in patent publications WO2008062460 and U.S. Pat. No. 6,046,353 and is shown in scheme 3. This involves condensation of diethyl malonate with isovaleraldehyde followed by cyanation. The product is selectively hydrolyzed to cyano ester which on hydrolysis gave cyano acid. The cyano acid was hydrogenated to racemic Pregabalin. Finally it was resolved by using (S)-mandalic acid with overall yield of 15.5% and ee>99.5% over 6 steps.

Another commonly used scaffold was found to be 3-isobutylglutaric acid anhydride (IBG). In US Patent publication No. 20090143615 and European Patent publication EP2067768 describes synthesis of Pregabalin as shown in scheme 4 that involves the ring opening by hydrazine followed by conversion to urethane acid by Curtius rearrangement. This intermediate was resolved using (S)-PEA. Release of chiral auxiliary followed by hydrolysis gave Pregabalin in overall yield of 12% over 4 steps with 99.8% ee. This method also suffers from the loss of unwanted R-isomer which can not be efficiently recycled.

Asymmetric ring opening of IBG and subsequent chemical transformation to enantiopure Pregabalin constitute another approach. The Patent Publication WO2008118427 describes the synthesis of (S)-Pregabalin depicted in scheme 5, starting from 3-isobutyl-glutaric anhydride that involves the stereo selective ring opening with (S)-PEA with good yield and ee purity. This was converted to amide by mixed anhydride approach. The amide was subjected to Hoffman degradation followed by PEA amide hydrolysis in two different approach to form Pregabalin in 59.5 and 38.8% respectively with purity >99.5%.

In a similar fashion, US Patent publication US 20070293694 described stereoselective ring opening of IBG with methanol in presence of molar equivalent of quinidine with high yield, however the ee is not satisfactory. Subsequent steps involve amidation followed by Hoffmann degradation. Use of molar equivalent of quinidine (expensive) and low ee makes the process unattractive.

In yet another approach similar to that described in scheme 5, Patent publication WO2007035789 described stereo selective ring opening of 3-isobutyl-glutaric anhydride (IBG) with (R)-PEA as described in scheme 7. The chiral auxiliary was replaced by amide using alkali metal amide at low temperature followed by Hoffmann degradation to give (S)-Pregabalin in overall 32.9% yield in three steps.

The synthesis reported in Patent publication WO2009081208 is shown in scheme 8. The ketone was converted to β-keto ester derivative which was converted to (S)-β-hydroxy intermediate by two different ways. The first method involve mauri yeast catalyzed reduction of ketone to give 50% yield and 99% ee while the second involve hydrogenation with [(S)—Ru (BINAP)Cl2]2.NEt3 (0.00046 eq=0.44% w/w) in 66% yield and >99% ee. Another key step is the conversion of alcohol to inverted bromo using Br2—PPh3 and also involves chromatography. The bromo compound is again completely inverted to (S) configuration with nitromethane and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Hydrolysis of ester and hydrogenation of nitro completes the synthesis with an overall yield of 13% and ee>99% involving 6 steps.

Another enzymatic route is shown in scheme 9 for the synthesis of Pregabalin was described in Patent publication US 20100204503. This involves kinetic resolution by lipase as the key step. The condensation of isovaleraldehyde with ethyl cyanoacetate followed by cyanation gave racemic dicyano compound. The Nitilase enzyme was used to get (S)-cyano acid and unwanted dinitrile which was racemized with DBU in toluene for recycling. The hydrogenation of (S)-tert butyl amine salt cyano acid gave (S)-Prgabalin in 7.7% overall yield with ˜100% ee over 4 steps. The extremely poor yield at the final stage makes this process economically unviable.

A rather more efficient enzymatic route is shown in scheme 10 was described in Patent publication US 2005028302. In this process the cyano diacid diethyl ester was enzymatically hydrolyzed to (S)-cyano ester monoacid potassium salt and the unwanted isomer was racemized. The salt was either reduced to a lactam acid followed by hydrolytic decarboxylation to Pregabalin with 34% overall yield and over 3 steps with ee>99.5%. Alternatively the (S)-cyano ester monoacid potassium salt was converted to cyano monoacid potassium salt that was hydrogenated to Pregabalin in 30% overall yield over 3 steps with 99.75% ee. Although it looks a reasonably good process however space vs. time yield will not be cost effective.

Finally there are some reports on asymmetric synthesis of Pregabalin mostly of academic interest due to the fact that they either involve longer sequence or provide Pregabalin with low ee. The scheme reported in J. Org Chem., 2003, 68, 5731-34 as shown in scheme 11, which described Bayllis-Hillman condensation and subsequent carbonate formation with chloroformate. The carbonate was subjected to CO insertion. The conjugated nitrile was hydrolyzed and converted to tert-butyamine salt that was stereo selectively hydrogenated to cyano acid using [(R,R)-(Me-DuPHOS)Rh(COD)].BF4, followed by hydrogenation of CN with Ni to give Pregabalin in 41.5% overall yield with ee 99.8% over 6 steps.

The synthesis described in Org. Lett., 2007, 9, 5307-09 as shown in scheme 12 involves asymmetric Micheal addition of nitromethane to αβ-unsaturated aldehyde using chiral catalyst. This catalyst need to be prepared from D-proline that involve 5 steps The number of steps are only three however ee is on the lower side. Additional resolution will be required Therefore this approach can not be economically viable.

Based on the drawbacks mentioned in all the prior arts above, accordingly therefore, there is an urgent need to develop a process for the preparation of a compound of formula (I), which is readily amenable to scale-up. Hence, we focused our research to simplify the process for the preparation of a compound of formula (I) with greater yield, higher chemical and chiral purity by using a genetically modified nitralase enzyme as a biocatalyst in a substantially cost effective and eco-friendly manner and to obviate the problems associated with the prior art process(s).